Introduction
Aglaonema Red Siam is one of the most beautiful ornamental plants, admired for its bright red and green foliage. It belongs to the Araceae family and is a favorite choice for indoor decoration due to its vibrant colors and low maintenance.
However, conventional propagation methods like stem cuttings are slow and inefficient for large-scale production. Therefore, Plant Tissue Culture (PTC) provides an advanced and rapid method for mass propagation of Aglaonema Red Siam. Through tissue culture, hundreds of genetically identical and disease-free plants can be produced from a small piece of plant tissue (explant) in a controlled, sterile environment.
Explant Used
| Type of Explant | Description |
|---|---|
| Shoot Tips | Actively growing regions of the plant used for rapid multiplication. |
| Stem Nodes | Contain axillary buds that can form new shoots. |
STAGES OF Tissue Culture :-
1. Initiation – A small pieces of plant tissue (explant) is collected from mother plant. Then sterilized explant to prevent contamination.
2. Establishment – It is placed on a nutrient medium containing essential nutrients, sugars, and hormones.
3. Multiplication – The explant develops into callus or shoots.
4. Differentiation – callus develop into specialized cells, tissues, or organs.
5. Shooting – Shoots develop from differentiation cells.
6. Rooting – Roots develop from shoots.
7. Hardening Stage – Roots plantlets gradually show external conditions like Light, Temperature, Humidity Then placed greenhouse or nursery.
Material Required :
| Petri Plates | Tissue paper |
| Test Tube | Distilled water |
| Test Tube Stand | Sterile Distilled water |
| Beaker | Sterile Surgical Blades |
| Culture Bottle | Autoclave Bag |
| Scissors | Filter paper |
| Spatula | Forecep |
| Scalpel | Conical flask |
| Cotton plug | Measuring Cylinder |
CHEMICALS FOR MS MEDIA:-
| Macronutrients |
| Micronutrients |
| Vitamines |
| Hormones |
| Agar powder |
| Iron source |
| Sucrose |
| NaOH |
| HCl |
CHEMICALS USE FOR STERILIZATION:-
| Ethanol | Bavistin |
| Savlon ( Dettol) | Antibiotic |
| Tween 20 (liquid soap) | Sodium hypochlorite |
PREPRATION OF MACRONUTRIENTS SOLUTION:-
| Macro nutrients | Original Concentration | Amount of salt after multiplying original concentration by 20X, Solution Volume (250ml) |
| NH4NO3 | 1650mg/l | 8250mg |
| KNO3 | 1900mg/l | 9500mg |
| KH2PO4 | 170mg/l | 850mg |
| MgSO4.7H2O | 370mg/l | 1850mg |
| CaCl2.2H2O | 440mg/l | 2200mg |
| CaCl2(fused) | 330mg/l | 1650mg |
PREPARATION OF MICRONUTRIENTS SOLUTION:-
| Micronutrients | Original Concentration | Amount of salt after multiplying original concentration by 100X, Solution Volume (250ml) |
| H3BO3 | 6.2mg/l | 155mg |
| MnSO4.4H2O | 22.3mg/l | 557.5mg |
| ZnSO4.7H2O | 8.6mg/l | 215mg |
| Na2MoO4.2H2O | 0.25mg/l | 6.25mg |
| CuSO4.5H2O | 0.025mg/l | 0.625mg |
| CoCl2.6H2O | 0.025mg/l | 0.625mg |
| Kl | 0.83mg/l | 20.75mg |
PREPARATION OF VITAMINS:-
| Vitamins | Original Concentration | Original Concentration multiplying by 1000X, Final solution volume (50ml) |
| Thiamine HCl | 0.1mg/l | 5mg |
| Pyridoxin HCl | 0.5mg/l | 25mg |
| Niacin | 0.5mg/l | 25mg |
| Glycine | 2.0mg/l | 100mg |
IRON SOURCES:-
| Sources | Original Concentration | Original Concentration multiplying by 400X, Final solution volume (50ml) |
| FeSO4.7H2O | 27.8mg/l | 2780mg |
| Na2EDTA.2H2O | 37.3mg/l | 3730mg |
MESOINSITOL:-
When we prepare 1 litre ms media then we will take 100mg mesoinositol.
SUCROSE (3%):-
When we prepare 1 litre ms media then we will take 30gm sucrose.
Preparation of ms (Murashige and Skoog ) media:-
| Materials | Final Volume (500ml) | Final Volume (250ml) |
| Distilled water | 250ml | 100ml |
| 20X, Macronutrients | 25ml | 12.5ml |
| 100X, Micronutrients | 1.25ml | 0.625ml |
| 1000X, Vitamins | 1.25ml | 0.625ml |
| 400X, Iron sources | 0.5ml | 0.25ml |
| Mesoinositol | 50mg | 25mg |
| Sucrose | 15gm | 7.5gm |
| Agar powder | 4gm | 2gm |
PREPARATION OF HORMONES:-
(Cytokinin & Auxin Solution Preparation)
BAP Stock:-100mg BAP in conical flask (250ml) dissolve with ethanol or 1M NaOH solution and BAP powder dissolve completely in solution and make final volume 100ml solution with Distilled water.
IBA Stock:- 100mg of IBA in conical flask (250) dissolve with ethanol or 1M NaOH solution and IBA powder dissolve completely in solution and make final volume to 100ml with Distilled water.
pH CHECK:- Prepare solution media pH range is 5.8 – 5.9 by using HCl or 1M NaOH.
Agar:- Take 8 gm agar powder and add in media ,mix with glass rod then in microwave the media for 14 minutes and agar dissolve completely in media.
STERILIZATION PROCESS :-
• First, we have cleaned glasswares such as test tube, Petri plates, conical flask, beaker, measuring cylinder, forecep, spatula , scalpel, surgical blades etc.Then, the cleaned items were dried in a hot air oven at180°C for 10 to 12 hours to ensure no water droplets remained. Then we have wrapped glassware in autoclave bag then put in autoclave with distilled water and culture media.
• Autoclave work at high pressure and temperature ( 15 psi, 121°C) for 15 to 20 minutes and kill the microorganisms. After sterilization removed glassware from sterilizer. And placed in laminar air flow then when we use it, we will turn on UV – ray light for 1 hour.
METHODOLOGY :
27 Dec 2025 – 30 Dec 2025
1. Select mother plants ( Shoot tips, Nodal part ) that are young shoot tips disease-free, and have high-yield and desirable traits.
2. Cut the mother plant in desirable shape and placed dry tray.
3. Wash the cut explant in running water for 15 minutes.
4. Fungicide Treatment (Bavistin):
Prepare Bavistin solution by dissolving 3 g Bavistin in 500 mL distilled water.
Soak explants in this solution for 20–25 minutes to eliminate fungal spores.
→ Bavistin acts as a systemic fungicide that prevents fungal contamination during tissue culture.
5. Handle the plant material with sterile gloves.
6. Then prepare total volume 300ml soap solution (30ml soap liquid and 270ml distilled water).
7. Wear glove hand and wash the plant material for 5 minutes.
7.Then remove the soap solution and placed in glass beaker.
8. Cut the material desirable size with the help of knife.
9. Then soak the material in savlon solution ( 3ml savlon and 300 ml distilled water) for 15 minutes.
10. Then wash the plant material from sterile distilled water 3 to 5 time in bottle beaker.
11. Prepare 4% sodium hypochlorite solution and taking sodium hypochlorite solution into a laminar air flow and soak the plant material in sodium hypochlorite solution for 20 minutes( for 1 jar 10ml sodium hypochlorite and 250ml sterile distilled water).
12. After 20 minutes then remove plant material from sodium hypochlorite solution and wash the plant material 3 to 5 times in sterile distilled water.
13. Then cut the plant material required culture bottle size with the help of knife on sterilized brown paper.
14. Prepare antibiotic solution using Cefotaxime antibiotic( 1ml antibiotic and 200ml sterile distilled water) and soak the plant material in this solution for 30 minutes. Using only fresh antibiotic solution.
15. Then inoculate the plant material in culture medium( bottle beaker) and remove laminar air flow placed inoculate culture bottle at 24°C with photo periods of 6 to 7 hours light and 8 hours darkness for tissue culture sterile environment.
31 Dec 2025
Preparation Of vitamin Stock for MS Media :
| Vitamins | Original Concentration | Original Concentration multiplying by 1000X, Final solution volume (50ml) |
| Thiamine HCl | 0.1mg/l | 5mg |
| Pyridoxin HCl | 0.5mg/l | 25mg |
| Niacin | 0.5mg/l | 25mg |
| Glycine | 2.0mg/l | 100mg |
Preparation Of Iron Source :
| Sources | Original Concentration | Original Concentration multiplying by 400X, Final solution volume (50ml) |
| FeSO4.7H2O | 27.8mg/l | 2780mg |
| Na2EDTA.2H2O | 37.3mg/l | 3730mg |
